Lipid Peroxidation-induced DNA Damage Research Articles

INCREASED URINARY 8-OXO-7,8-DIHYDRO-2′-DEOXYGUANOSINE EXCRETION IN A SAMPLE OF EGYPTIAN CHILDREN WITH BETA THALASSEMIA MAJOR: MARKER FOR LIPID PEROXIDATION-INDUCED DNA DAMAGE

Objective: The objective of this research was to evaluate oxidative stress status in children with β-thalassemia major.Methods: Our study was conducted in children with β-thalassemia aged from 5 to 15 years. Investigate the urinary excretion of human 8-oxo-7,8- dihydro-2′-deoxyguanosine, which will be analyzed by enzyme-linked immunosorbent assay. To investigate serum levels of antioxidant enzymes include glutathione s-transferase (GST) and catalase (CAT).Results: We found a significant elevation of the urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine level with p=0.001 compared to control group, a significant reduction of both GST and CAT p=0.05 and 0.03, respectively, compared to control group. There was a significant negative correlation between urinary 8-oxo-7,8-dihydro-2′-deoxyguanisine and CAT level, r=−0.378, p=0.016, hemoglobin - r=−0.610, p=0.001, hematocrit (%) - r=−0.478, p=0.002, while a significant positive correlation between urinary 8-oxo-7,8-dihydro-2′-deoxyguanisine and alanine aminotransferase - r=0.547, p=0.001, and serum ferritin - r=0.391, p=0.013. There was a significant negative correlation between CAT and serum ferritin - r=−0.320, p=0.44.Conclusion: We conclude that the strongly increased urinary excretion 8-oxo-7,8=dihydro-2′-deoxyguanisine indicates elevated lipid peroxidation induced DNA damage in internal organs such as the liver. These highly pro mutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.

Increased levels of urinary biomarkers of lipid peroxidation products among workers occupationally exposed to diesel engine exhaust

Diesel engine exhaust (DEE) was found to induce lipid peroxidation (LPO) in animal exposure studies. LPO is a class of oxidative stress and can be reflected by detecting the levels of its production, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and etheno-DNA adducts including 1,N6-etheno-2′-deoxyadenosine (ɛdA) and 3,N4-etheno-2′-deoxycytidine (ɛdC). However, the impact of DEE exposure on LPO has not been explored in humans. In this study, we evaluated urinary MDA, 4-HNE, ɛdA, and ɛdC levels as biomarkers of LPO among 108 workers with exclusive exposure to DEE and 109 non-DEE-exposed workers. Results showed that increased levels of urinary MDA and ɛdA were observed in subjects occupationally exposed to DEE before and after age, body mass index (BMI), smoking status, and alcohol use were adjusted (all p < 0.001). There was a statistically significant relationship between the internal exposure dose (urinary ΣOH-PAHs) and MDA, 4-HNE, and ɛdA (all p < 0.001). Furthermore, significant increased relations between urinary etheno-DNA adduct and MDA, 4-HNE were observed (all p < 0.05). The findings of this study suggested that the level of LPO products (MDA and ɛdA) was increased in DEE-exposed workers, and urinary MDA and ɛdA might be feasible biomarkers for DEE exposure. LPO induced DNA damage might be involved and further motivated the genomic instability could be one of the pathogeneses of cancer induced by DEE-exposure. However, additional investigations should be performed to understand these observations.

Slow repair of lipid peroxidation-induced DNA damage at p53 mutation hotspots in human cells caused by low turnover of a DNA glycosylase.

Repair of oxidative stress- and inflammation-induced DNA lesions by the base excision repair (BER) pathway prevents mutation, a form of genomic instability which is often observed in cancer as ‘mutation hotspots’. This suggests that some sequences have inherent mutability, possibly due to sequence-related differences in repair. This study has explored intrinsic mutability as a consequence of sequence-specific repair of lipid peroxidation-induced DNA adduct, 1, N6-ethenoadenine (εA). For the first time, we observed significant delay in repair of ϵA at mutation hotspots in the tumor suppressor gene p53 compared to non-hotspots in live human hepatocytes and endothelial cells using an in-cell real time PCR-based method. In-cell and in vitro mechanism studies revealed that this delay in repair was due to inefficient turnover of N-methylpurine-DNA glycosylase (MPG), which initiates BER of εA. We determined that the product dissociation rate of MPG at the hotspot codons was ≈5–12-fold lower than the non-hotspots, suggesting a previously unknown mechanism for slower repair at mutation hotspots and implicating sequence-related variability of DNA repair efficiency to be responsible for mutation hotspot signatures.

Aberrant repair of etheno–DNA adducts in leukocytes and colon tissue of colon cancer patients

To assess the role of lipid peroxidation-induced DNA damage and repair in colon carcinogenesis, the excision rates and levels of 1, N 6-etheno-2′-deoxyadenosine (εdA), 3, N 4-etheno-2′-deoxycytidine (εdC), and 1, N 2-etheno-2′-deoxyguanosine (1, N 2-εdG) were analyzed in polymorphic blood leukocytes (PBL) and resected colon tissues of 54 colorectal carcinoma (CRC) patients and PBL of 56 healthy individuals. In PBL the excision rates of 1, N 6-ethenoadenine (εAde) and 3, N 4-ethenocytosine (εCyt), measured by the nicking of oligodeoxynucleotide duplexes with single lesions, and unexpectedly also the levels of εdA and 1, N 2-εdG, measured by LC/MS/MS, were lower in CRC patients than in controls. In contrast the mRNA levels of repair enzymes, alkylpurine- and thymine-DNA glycosylases and abasic site endonuclease (APE1), were higher in PBL of CRC patients than in those of controls, as measured by QPCR. In the target colon tissues εAde and εCyt excision rates were higher, whereas the εdA and εdC levels in DNA, measured by 32P-postlabeling, were lower in tumor than in adjacent colon tissue, although a higher mRNA level was observed only for APE1. This suggests that during the onset of carcinogenesis, etheno adduct repair in the colon seems to be under a complex transcriptional and posttranscriptional control, whereby deregulation may act as a driving force for malignancy.

Increased urinary 1, N6-ethenodeoxyadenosine and 3, N4-ethenodeoxycytidine excretion in thalassemia patients: Markers for lipid peroxidation-induced DNA damage

Thalassemic diseases including homozygous β-thalassemia and β-thalassemia/Hb E (β-Thal/Hb E) are prevalent in Southeast Asia. Iron overload is a common complication in β-thalassemia patients which induces intracellular oxidative stress and lipid peroxidation (LPO). LPO end products generate miscoding etheno adducts in DNA which after their repair are excreted in urine. We investigated whether urinary levels of 1, N 6 -ethenodeoxyadenosine (εdA) and 3, N 4 -ethenodeoxycytidine (εdC) can serve as putative cancer risk markers in β-Thal/Hb E patients. εdA and εdC levels were assayed in collected urine samples by immunoprecipitation-HPLC-fluorescence and 32P-postlabeling TLC, respectively. Mean εdA (fmol/µmol creatinine) levels in urine of β-Thal/Hb E patients ranged from 4.8 to 120.4 (33.8 ± 3.9; n = 37) and were 8.7 times higher compared to asymptomatic controls (1.4–13.8; 3.9 ± 0.8; n = 20). The respective εdC levels ranged from 0.15 to 32.5 (5.2 ± 1.3; n = 37) and were increased some 13 times over controls (0.04–1.2; 0.4 ± 0.7; n = 20). εdC levels were correlated positively with NTBI ( r = 0.517; P = 0.002), whereas εdA showed only a trend ( r = 0.257; P = 0.124). We conclude that the strongly increased urinary excretion of etheno adducts indicates elevated LPO-induced DNA damage in internal organs such as the liver. These highly promutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.

Decreased levels of lipid peroxidation-induced DNA damage in the onset of atherogenesis in apolipoprotein E deficient mice

Increased oxidative stress and subsequent lipid peroxidation (LPO) are thought to be critical events in the formation of atherosclerotic lesions in apolipoprotein E deficient mice (ApoE-KO). LPO derived reactive aldehydes react with DNA to form exocyclic etheno-DNA adducts. These pro-mutagenic DNA lesions are known to be involved in the initiation of carcinogenesis, but their role in the development of atherosclerosis is unknown. In the present study we show that levels of the LPO derived 1, N 6-ethenodeoxyadenosine (ɛdA) and 3, N 4-ethenodeoxycytidine (ɛdC) were both significantly lower in aorta of 12 weeks old ApoE-KO mice as compared to their wild type controls (1.6 ± 0.3 versus 3.2 ± 0.8 ɛdA per 10 8 parent nucleotides, P = 0.04 and 4.8 ± 0.8 versus 9.2 ± 2.1 for ɛdC, P = 0.02). Moreover, levels of both DNA adduct types were inversely related with total plasma cholesterol levels. Consequently, lowest etheno-DNA adduct levels were observed in ApoE-KO mice on a high fat diet. Hypercholesterolemia has previously been associated with increased expression of base excision repair (BER) enzymes, which could explain the lower levels of etheno-DNA adducts in ApoE-KO mice as compared to wild type controls. Indeed, increased staining for the BER-specific DNA repair enzyme apurinic/apyrimidinic endonuclease (Ape1/Ref1) was observed by immunohistochemistry in the endothelium and the first layers of arterial smooth muscle cells of ApoE-KO mice as compared to their wild type counterparts. A high fat diet further increased overall Ape1/Ref1 protein expression in ApoE-KO mice. Although these data suggest no role for increased LPO derived DNA damage in the onset of atherogenesis in ApoE-KO mice, the potentially modulating role of Ape1/Ref1 in the arterial wall deserves further attention.

Blood Pressure Control Goes Nuclear

See related article, pages 902–910 The control of blood pressure is regulated with extreme precision, and requires integration of information from the central nervous system, the kidneys, the vasculature, and the heart. Salt and water balances, sympathetic activity, arterial stiffness, and the tone of resistance vessels determine the chronic regulation of intravascular pressure. It is a standard experiment for physicians-in-training to manipulate these activities using classical physiological techniques and observe the homeostatic mechanisms that return blood pressure to its normal levels. Given the many controls that exist to maintain blood pressure within a narrow and appropriate range, it is all the more remarkable that clinical hypertension is as common as it is, affecting more than 50 million Americans and 75% of the population older than 65 years of age. The paracrine and endocrine mechanisms that regulate blood pressure are now reasonably well understood, and indeed provide the pharmacological basis for most antihypertensive therapies. In contrast, the intracellular events that tune blood pressure are less well characterized. In particular, nuclear events that control blood pressure are not well known. This is a major gap in our understanding of the problem insofar as long-term changes in gene regulation are a likely proximate cause of the systematic events that result in chronic hypertension. A report in this issue of Circulation Research by Irani and colleagues,1 which clearly defines a role for the multifunctional nuclear protein apurinic/apyrimidinic endonuclease (APE1, also known as redox factor-1) in chronic regulation of vascular tone in vivo, represents an important step toward understanding how nuclear proteins may integrate diverse signals to tune the genetic program that regulates blood pressure. APE1 is a multifunctional protein (Figure, A). It contains tandem cysteine residues within its amino terminus that exert a reducing activity that is responsible for regulating redox-sensitive …

A modified immuno-enriched 32P-postlabeling method for analyzing the malondialdehyde-deoxyguanosine adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)- pyrimido[1,2-alpha]purin-10(3H)one in human tissue samples.

The malondialdehyde-modified DNA adduct, 3-(2-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)one (M1dG) has been detected in human tissues and is considered to be a promising biomarker for estimating lipid peroxidation-induced DNA damage. With the aim to analyze the M1dG in small amounts of DNA (<10 microg) and to improve the sensitivity, we have developed an immuno-enriched 32P-postlabeling HPLC method. The main modifications included the following steps: (i) an optimization of the immunoenrichment conditions using a monoclonal antibody (MAb D 10A1), (ii) a single labeling step of the purified M1dG 3'-monophosphate to its 5'-monophosphate at pH 6.8, (iii) the addition of O4-ethylthymidine 3'-monophosphate as an internal standard, and (iv) a prepurification of the labeled adduct on a polyethyleneimine minicolumn before HPLC analysis. With this protocol, the percent recovery of M1dG was found to be approximately 70 +/- 20; the detection limit in biological samples was approximately 200 amol M1dG from 10 microg of DNA, corresponding to 6 adducts/10(9) nucleotides. In conclusion, our modified method shows a high sensitivity and specificity; when applied to human breast and liver tissue samples, background levels of the M1dG could be reproducibly detected. This ultrasensitive detection method is thus suitable for applications in human biomonitoring and molecular epidemiology studies.

Benzo[a]pyrene Enhances Lipid Peroxidation Induced DNA Damage in Aorta of Apolipoprotein E Knockout Mice

The genotoxic compound benzo[a]pyrene (B[a]P) enhances atherosclerotic plaque progression, possibly by inducing oxidative stress and subsequent lipid peroxidation (LPO). Since LPO plays a key role in atherosclerosis, stable LPO derived DNA modifications such as 1,N6-ethenodeoxyadenosine (εdA) and 3,N4-ethenodeoxy-cytidine (εdC) may be useful biomarkers for in vivo oxidative stress. In this study, benzo[a]pyrene-diol-epoxide (BPDE)-DNA, εdA and εdC were determined by 32P-postlabelling in apolipoprotein E knockout (ApoE-KO) mice treated with 5 mg/kg B[a]P by gavage. After 4 days, BPDE-DNA adduct levels were higher in aorta (10.8±1.4 adducts/108 nucleotides) than in lung (3.3±0.7, P<0.05), which is a known target organ for B[a]P. Levels of εdA were higher in aorta of B[a]P-exposed animals than in unexposed controls (8.1±4.4 vs 3.4±2.1 adducts per 108 parent nucleotides, P<0.05). On the other hand, εdC levels were not affected by B[a]P exposure. Serum low density lipoprotein (LDL) levels were lower in B[a]P-exposed mice than in controls (9.3±3.7 and 13.3±4.0 mmol/l, respectively), whereas high density lipoprotein (HDL) levels were higher (1.4±1.6 and 0.4±0.3 mmol/l, respectively). Consequently, a three-fold difference in the LDL/HDL ratio was observed (P=0.001). εdA levels were positively related with plasma HDL concentrations (R=0.68, P=0.02), suggesting that the HDL mediated protection of the vessel wall against reactive lipid peroxides was reduced in B[a]P-exposed apoE-KO mice. Our observations show that direct as well as lipid peroxidation induced DNA damage is formed by B[a]P in aorta of apoE-KO mice, which may be involved in atherosclerotic plaque progression. This study further indicates that etheno-DNA adducts are useful biomarkers for in vivo oxidative stress in atherosclerosis.

Exocyclic DNA adducts as oxidative stress markers in colon carcinogenesis: potential role of lipid peroxidation, dietary fat and antioxidants.

Molecular pathways to colorectal cancer involve multiple genetic changes, whereby extensive oxyradical damage causes mutations in cancer-related genes and leads to a cycle of cell death and regeneration. Besides direct oxidative DNA-damage, reactive oxygen and nitrogen species can induce etheno (epsilon)-DNA adducts mainly via trans-4-hydroxy-2-nonenal, generated as the major aldehyde by lipid peroxidation (LPO) of omega-6 PUFAs. Patients with familial adenomatous polyposis (FAP) develop multiple colorectal adenomas. In affected tissues increased LPO could be triggered due to increased arachidonic acid metabolism as a result of elevated cyclooxygenases. Our studies demonstrated an increased epsilon-DNA adduct level in affected colon epithelia of FAP patients. Epsilon-DNA adducts are promutagenic and can cause genomic instability that drives colorectal adenoma to malignancy. We have further investigated the potential chemopreventive properties of olive oil and its polyphenolic components. 'Mediterranean diet', of which olive oil is a major fatty acid source, has protective effects against human breast and colorectal cancers. Olive oil extracts and the newly identified lignan fractions showed high antioxidant capacity in vitro. As epsilon-DNA adducts are biomarkers for oxidative stress and LPO induced DNA damage, they can verify the efficacy of newly identified antioxidants, e.g. from olive oil, as chemopreventive agents against colon carcinogenesis.

Immunohistochemical detection of 1,N(6)-ethenodeoxyadenosine, a promutagenic DNA adduct, in liver of rats exposed to vinyl chloride or an iron overload.

Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N(6)-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed approximately 1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride-treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno-DNA adduct that may play a role in oxidative stress and lipid peroxidation-induced DNA damage in carcinogenesis.

Lipid peroxidation induced DNA damage: Role in carcinogenesis

Lipid peroxidation induced DNA damage: Role in carcinogenesis